Paradoxical development of polymyositis-like autoimmunity through augmented expression of autoimmune regulator (AIRE).

Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.

Transient suppression of late-stage neuronal progenitor cell differentiation in the hippocampal dentate gyrus of rat offspring after maternal exposure to nicotine.

To examine the developmental exposure effect of nicotine (NIC) on hippocampal neurogenesis, pregnant Sprague-Dawley rats were treated with (-)-NIC hydrogen tartrate salt through drinking water at 2, 10 or 50 ppm from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, immunohistochemically doublecortin (Dcx)(+) cells increased at ≥10 ppm in the dentate subgranular zone (SGZ) as examined in male offspring; however, dihydropyrimidinase-like 3 (TUC4)(+) cells decreased at 2 ppm, and T box brain 2 (Tbr2)(+) cells were unchanged at any dose. Double immunohistochemistry revealed decreases in TUC4(+)/Dcx(+) and TUC4(+)/Dcx(-) cells, an increase in TUC4(-)/Dcx(+) cells at 2 and 10 ppm and an increase in Tbr2(-)/Dcx(+) cells at 50 ppm, suggesting an increase in type-3 progenitor cells at ≥2 ppm and decrease in immature granule cells at 2 and 10 ppm. The number of mature neuron-specific NeuN(-) progenitor cells expressing nicotinic acetylcholine receptor α7 in the SGZ and mRNA levels of Chrna7 and Chrnb2 in the dentate gyrus was unchanged at any dose, suggesting a lack of direct nicotinic stimulation on progenitor cells. In the dentate hilus, glutamic acid decarboxylase 67(+) interneurons increased at ≥10 ppm. All changes disappeared on PND 77. Therefore, maternal exposure to NIC reversibly affects hippocampal neurogenesis targeting late-stage differentiation in rat offspring. An increase in interneurons suggested that their activation affected granule cell differentiation. The lowest observed adverse effect level was at 2 ppm (0.091 mg/kg/day as a free base) by the affection of hippocampal neurogenesis at ≥2 ppm.

Apolipoprotein A-II suppressed concanavalin A-induced hepatitis via the inhibition of CD4 T cell function.

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

Expression patterns of cell cycle proteins in the livers of rats treated with hepatocarcinogens for 28 days.

Some hepatocarcinogens induce cytomegaly, which reflects aberrant cell cycling and increased ploidy, from the early stages of administration to animals. To clarify the regulatory molecular mechanisms behind cell cycle aberrations related to the early stages of hepatocarcinogenesis, we performed gene expression analysis using microarrays and real-time reverse transcription polymerase chain reaction followed by immunohistochemical analysis in the livers of rats treated with the cytomegaly inducing hepatocarcinogens thioacetamide (TAA), fenbendazole, and methyleugenol, the cytomegaly non-inducing hepatocarcinogen piperonyl butoxide (PBO), or the non-carcinogenic hepatotoxicants acetaminophen and α-naphthyl isothiocyanate, for 28 days. Gene expression profiling showed that cell cycle-related genes, especially those of G(2)/M phase, were mostly upregulated after TAA treatment. Immunohistochemical analysis was performed on cell cycle proteins that were upregulated by TAA treatment and on related proteins. All hepatocarcinogens, irrespective of their cytomegaly inducing potential, increased liver cells immunoreactive for p21(Cip1), which acts on cells arrested in G(1) phase, and for Aurora B or Incenp, which is suggestive of an increase in a cell population with chromosomal instability caused by overexpression. PBO did not induce cell proliferation after 28-day treatment. Hepatocarcinogens that induced cell proliferation after 28-day treatment also caused an increase in p53(+) cells in parallel with increased apoptotic cells, as well as increased population of cells expressing M phase-related proteins nuclear Cdc2, phospho-Histone H3, and HP1α. These results suggest that hepatocarcinogens may increase cellular populations arrested in G1 phase or showing chromosomal instability after 28-day treatment. Hepatocarcinogens that induce cell cycle facilitation may cause M phase arrest accompanied by apoptosis.

Cellular distribution of cell cycle-related molecules in the renal tubules of rats treated with renal carcinogens for 28 days: relationship between cell cycle aberration and carcinogenesis.

Some renal carcinogens can induce karyomegaly, which reflects aberrant cell division in the renal tubules, from the early stages of exposure. To clarify the cell cycle-related changes during the early stages of renal carcinogenesis, we performed immunohistochemical analysis of tubular cells in male F344 rats treated with carcinogenic doses of representative renal carcinogens for 28 days. For this purpose, the karyomegaly-inducing carcinogens ochratoxin A (OTA), ferric nitrilotriacetic acid, and monuron, and the non-karyomegaly-inducing carcinogens tris(2-chloroethyl) phosphate and potassium bromate were examined. For comparison, a karyomegaly-inducing non-carcinogen, p-nitrobenzoic acid, and a non-carcinogenic non-karyomegaly-inducing renal toxicant, acetaminophen, were also examined. The outer stripe of the outer medulla (OSOM) and the cortex + OSOM were subjected to morphometric analysis of immunoreactive proximal tubular cells. Renal carcinogens, irrespective of their karyomegaly-inducing potential, increased proximal tubular cell proliferation accompanied by an increase in topoisomerase IIα-immunoreactive cells, suggesting a reflection of cell proliferation. Karyomegaly-inducing carcinogens increased nuclear Cdc2-, γH2AX-, and phosphorylated Chk2-immunoreactive cells in both areas, the former two acting in response to DNA damage and the latter one suggestive of sustained G2. OTA, an OSOM-targeting carcinogen, could easily be distinguished from untreated controls and non-carcinogens by evaluation of molecules responding to DNA damage and G2/M transition in the OSOM. Thus, all renal carcinogens examined facilitated proximal tubular proliferation by repeated short-term treatment. Among these, karyomegaly-inducing carcinogens may cause DNA damage and G2 arrest in the target tubular cells.

Reversible aberration of neurogenesis targeting late-stage progenitor cells in the hippocampal dentate gyrus of rat offspring after maternal exposure to acrylamide.

We have recently shown that maternal exposure to acrylamide (AA) impaired neurogenesis in rat offspring measured by the increase in interneurons producing reelin, a molecule regulating migration and correct positioning of developing neurons, in the hippocampal dentate gyrus. To clarify the cellular target of AA on hippocampal neurogenesis and its reversibility after maternal exposure, pregnant Sprague-Dawley rats were given drinking water containing AA at 0, 4, 20, 100 ppm on day 10 of pregnancy through day 21 after delivery on weaning. Male offspring were examined immunohistochemically on postnatal day (PND) 21 and PND 77. For comparison, male pups of direct AA-injection control during lactation (50 mg/kg body weight, intraperitoneally, 3 times/week) were also examined. On PND 21, maternal AA-exposure decreased progenitor cell proliferation in the subgranular zone (SGZ) from 20 ppm accompanied with increased density of reelin-producing interneurons and NeuN-expressing mature neurons within the hilus at 100 ppm, similar to the direct AA-injection control. In the SGZ examined at 100 ppm, cellular populations immunoexpressing doublecortin or dihydropyrimidinase-like 3, suggesting postmitotic immature granule cells, were decreased. On PND 77, the SGZ cell proliferation and reelin-producing interneuron density recovered, while the hilar mature neurons sustained to increase from 20 ppm, similar to the direct AA-injection control. Thus, developmental exposure to AA reversibly affects hippocampal neurogenesis targeting the proliferation of type-3 progenitor cells resulting in a decrease in immature granule cells in rats. A sustained increase in hilar mature neurons could be the signature of the developmental effect of AA.

Mechanistic study on liver tumor promoting effects of flutamide in rats.

Flutamide (FLU), a nonsteroidal anti-androgen, is used for the treatment of prostate cancer but is also a cytochrome P450 (CYP) 1A inducer. Some CYP1A inducers are known to exert hepatocellular tumor-promoting activities in rodents, and reactive oxygen species (ROS) produced by CYP1A1 induction via a metabolism of FLU is probably involved in the liver tumor promotion. In the present study, to clarify the possible liver tumor promoting effect of FLU, a two-stage liver carcinogenesis assay was performed using male F344 rats. Rats received an intraperitoneal (ip) injection of 200 mg/kg body weight of N-diethylnitrosamine (DEN) and fed a diet containing 0, 0.1 or 0.2% FLU for 6 weeks. After 2 weeks of DEN treatment, all rats were subjected to two-thirds partial hepatectomy. Animals were killed 8 weeks after ip injection of DEN. Immunohistochemically, the number and area of glutathione S-transferase placental form (GST-P)-positive foci significantly increased in the liver of rats given 0.2% FLU as compared with the control. Ki-67-positive cell ratio also increased in rats given FLU at both concentrations. ROS generation in the microsomal fraction and production of thiobarbituric acid-reactive substance [TBARS] and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in the liver did not increase in any of the FLU-treated groups. The results of microarray and real-time RT-PCR revealed that phase 1 drug-metabolizing enzymes such as CYP1A1, Ugt1a61 and Nqo1 and phase II drug-metabolizing enzymes such as Yc2, Akr1b7, Akr1b8, Akr1b10, Aldh1a1, Gpx2 and Me1 were up-regulated in rats treated with FLU. In addition, the MAPK pathway family-related genes such as Prkcα, Mek1, Rafb, Myc, Mek2, Raf1 and Egfr were also up-regulated in FLU-treated groups. The results of the present study indicate that FLU is a CYP1A inducer but does not cause any production of microsomal ROS in the liver and suggest that microsomal ROS is not involved in the liver tumor promoting effect of FLU.

Similar distribution changes of GABAergic interneuron subpopulations in contrast to the different impact on neurogenesis between developmental and adult-stage hypothyroidism in the hippocampal dentate gyrus in rats.

Hypothyroidism affects neurogenesis. The present study was performed to clarify the sensitivity of neurogenesis-related cellular responses in the hippocampal dentate gyrus between developmental and adult-stage hypothyroidism. An exposure study of methimazole (MMI) as an anti-thyroid agent at 0, 50, 200 ppm in the drinking water was performed using pregnant rats from gestation day 10 to postnatal day (PND) 21 (developmental hypothyroidism) and adult male rats by setting an identical exposure period from PND 46 through to PND 77 (adult-stage hypothyroidism). Offspring with developmental hypothyroidism were killed at PND 21 or PND 77, and animals with adult-stage hypothyroidism were killed at PND 77. Proliferation and apoptosis were unchanged in the dentate subgranular zone by either developmental or adult-stage hypothyroidism. With regard to precursor granule cells, a sustained reduction of paired box 6-positive stem or early progenitor cells and a transient reduction of doublecortin-positive late-stage progenitor cells were observed after developmental hypothyroidism with MMI at 50 and 200 ppm. These cells were unchanged by adult-stage hypothyroidism. With regard to γ-aminobutyric acid (GABA) ergic interneuron subpopulations in the dentate hilus, the number of parvalbumin-positive cells was decreased and the number of calretinin-positive cells was increased after both developmental and adult-stage hypothyroidism with MMI at 50 and 200 ppm. Fluctuations in GABAergic interneuron numbers with developmental hypothyroidism continued through to PND 77 with 200 ppm MMI. Considering the roles of GABAergic interneuron subpopulations in neurogenesis and neuronal differentiation, subpopulation changes in GABAergic interneurons by hypothyroidism may be the signature of aberrant neurogenesis even at the adult stage.

Transient aberration of neuronal development in the hippocampal dentate gyrus after developmental exposure to brominated flame retardants in rats.

We immunohistochemically investigated the impact and reversibility of three brominated flame retardants (BFRs) known to be weak thyroid hormone disruptors on neuronal development in the hippocampal formation and apoptosis in the dentate subgranular zone. Pregnant Sprague-Dawley rats were exposed to 10, 100, or 1,000 ppm decabromodiphenyl ether (DBDE); 100, 1,000 or 10,000 ppm tetrabromobisphenol A (TBBPA) or 1,2,5,6,9,10-hexabromocyclododecane (HBCD) in the diet from gestational day 10 through to day 20 after delivery (weaning). On postnatal day (PND) 20, interneurons in the dentate hilus-expressing reelin increased with all chemicals, suggestive of aberration of neuronal migration. However, this increase had disappeared by PND 77. NeuN-positive mature neurons increased in the hilus on PND 77 with all chemicals. In the subgranular zone on PND 20, an increase in apoptotic bodies suggestive of impaired neurogenesis was observed after exposure to TBBPA or HBCD. The effects on neuronal development were detected at doses of ≥100 ppm DBDE; ≥1,000 ppm TBBPA; and at least at 10,000 ppm HBCD. On PND 20, the highest dose of DBDE and HBCD revealed mild fluctuations in the serum concentrations of thyroid-related hormones suggestive of weak developmental hypothyroidism, while TBBPA did not. Thus, DBDE and TBBPA may exert direct effect on neuronal development in the brain, but hypothyroidism may be operated for DBDE and HBCD at high doses. An excess of mature neurons in the hilus at later stages may be the signature of the developmental effects of BFRs. However, the effect itself was reversible.

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database.

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificity in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing.

Evaluation of in vivo genotoxic potential of fenofibrate in rats subjected to two-week repeated oral administration.

Fenofibrate (FF), a peroxisome proliferator-activated receptor-alpha agonist, has been used as one of the hypolipidemic drugs in man and induces oxidative stress and promotes hepatocarcinogenesis in the liver of rodents. This chemical belongs to a class of non-genotoxic carcinogens, but DNA damage secondary to oxidative stress resulting from reactive oxygen species (ROS) generation is suspected in rodents given this chemical. To examine whether FF has genotoxic potential, partially hepatectomized F344 male rats were treated orally with 0, 1,000 or 2,000 mg/kg of FF for 2 weeks, followed by diet containing 0.15% 2 acetyl aminofluorene (2 AAF) for enhancement the tumor-promoting effect for 10 days and a single oral dose of carbon tetrachloride (CCl4) as the first experiment (liver initiation assay). As the second experiment, the in vivo liver comet assay was performed in hepatectomized rats, and the expression of some DNA repair genes was examined. In the liver initiation assay, the number and area of glutathione S-transferase placental form (GST-P)-positive single cells and foci did not increase in the FF treated groups. In the comet assay, positive results were obtained after 3 h of the last treatment of FF, and the expression of some DNA repair genes such as Apex1, Ogg1 and Mlh1 were upregulated in rats given the high dose of FF at 3 h after the treatment but not in 24 h after the treatment. The results of the present study suggest that FF causes some DNA damage in livers of rats, but is not a strong genotoxic substance leading to a DNA mutation since such DNA damage was repaired by the increased activity of some DNA repair genes.

Immunohistochemical analyses at the late stage of tumor promotion by oxfendazole in a rat hepatocarcinogenesis model.

The present study was performed to characterize immunohistochemically the expression levels of molecules related to not only xenobiotic and antioxidant functions but also cell proliferation and apoptosis in neoplastic lesions induced by the benzimidazole anthelmintic, oxfendazole (OX), at the late stage of its tumor promotion in a rat hepatocarcinogenesis model. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and 2 weeks later they were fed a diet containing 0% (basal diet) or 0.05% OX for 26 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and killed at week 28. Histopathologically, OX increased the incidence and multiplicity of altered foci (4.0- and 3.6-fold, respectively) and hepatocellular adenomas (HCAs) (3.0- and 5.5-fold, respectively). OX treatment induced 5.2- and 5.6-fold increases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and single-stranded DNA (ssDNA)-positive cells in HCAs compared with the surrounding tissue, respectively. Staining for the cell cycle regulators P21 and C/EBPα and the AhR-regulated CYP1A1 molecules decreased but increased reactivity of the Nrf2-regulated, detoxifing/antioxidant molecules aldo-keto reductase 7 (AKR7) and glutathione peroxidase 2 (GPX2) were also seen in HCAs compared with the surrounding hepatocytes. These results suggest that dysregulation of cell proliferation and apoptosis and escape from oxidative stress elicited by OX treatment play an important role in OX-induced hepatocarcinogenesis in rats.

Relationship between CYP1A induction by indole-3-carbinol or flutamide and liver tumor-promoting potential in rats.

To investigate liver tumor-promoting potentials of indole-3-carbinol (I3C) and flutamide (FLU), changes in mRNA expression of Cyp1a and genes encoding antioxidant/detoxifying enzymes in the liver, 6-week-old male F344 rats were subjected to medium-term liver bioassay. ß-Naphthoflavone (BNF), a strong CYP1A inducer, was also used for comparison. Two weeks after initiation with N-diethylnitrosamine (DEN), animals were fed a basal diet (untreated controls) or a diet containing 0.5% I3C, 0.1% FLU, or 0.5% BNF for 6 weeks. Each animal was subjected to a two-third partial hepatectomy 1 week after the start of promoter treatments. Histopathologically, I3C and BNF increased altered liver cell foci with the incidence (3.7- and 7.3-fold) and multiplicity (8.3- and 13.8-fold) compared with the DEN-alone group, respectively. Immunohistochemically, I3C significantly increased the number (3.1-fold; P < 0.01) and area (2.4-fold; P < 0.05) of foci positive for glutathione-S-transferase placental form (GST-P) compared with the DEN-alone group; FLU induced a slight but significant increase in the number of GST-P-positive foci (2.8-fold; P < 0.05) whereas BNF showed marked induction of the number and area of GST-P-positive foci (20- and 14-fold, respectively; P < 0.01). In parallel, I3C, FLU, and BNF markedly increased mRNA levels of Cyp1a1 (50-, 23-, 299-fold) and antioxidant/detoxifying enzymes such as Gpx2 and Nqo1 as shown by real-time reverse transcription-polymerase chain reaction analysis. These results suggest that I3C and FLU could promote hepatocellular tumors in parallel with that of CYP1A's potential to cause subsequent oxidative stress responses in rats.

Disruptive neuronal development by acrylamide in the hippocampal dentate hilus after developmental exposure in rats.

To examine whether developmental exposure to acrylamide (AA) impairs neuronal development, pregnant Sprague-Dawley rats were treated with AA at 0, 25, 50 or 100 ppm in drinking water from gestational day 6 until weaning on postnatal day 21. Offspring were immunohistochemically examined at the end of exposure. We investigated the expression of Reelin (a molecule regulating neuronal migration and positioning) in the hilus of the hippocampal dentate gyrus. As a positive control for direct exposure, AA (50 mg/kg body weight) was administered to pups by intraperitoneal injection 3 times per week during the lactation period. As well as pups directly injected with AA, maternally exposed offspring decreased body weight at 100 ppm; increased dose-dependently the number of Reelin-immunoreactive cells (from 25 ppm AA) and glutamic acid decarboxylase 67-immunoreactive cells (from 50 ppm AA), confirming an increase in γ-aminobutyric acid-ergic interneurons. We also noted decreased apoptosis in the neuroblast-producing subgranular zone of the dentate gyrus of maternally exposed pups at 100 ppm, as well as in directly AA-injected pups. These results suggest that a compensatory regulatory mechanism exists to correct impaired neurogenesis and mismigration caused by maternal exposure to AA during neuronal development. The lowest-observed-adverse-effect level of AA was determined to be 25 ppm (3.72 mg/kg body weight/day).

Suppressive effect of enzymatically modified isoquercitrin on phenobarbital-induced liver tumor promotion in rats.

To investigate the effect of enzymatically modified isoquercitrin (EMIQ) on hepatocellular tumor promotion induced by phenobarbital (PB), male rats were administered a single intraperitoneal injection of 200 mg/kg N-diethylnitrosamine (DEN) and then fed with a diet containing PB (500 ppm) for 8 weeks, with or without EMIQ (2,000 ppm) in the drinking water. One week after PB administration, rats underwent a two-thirds partial hepatectomy. The PB-induced increase in the number and area of glutathione S-transferase placental form-positive foci and the proliferating cell nuclear antigen-positive ratio was significantly suppressed by EMIQ. Real-time reverse transcription-polymerase chain reaction analysis revealed increases in mRNA expression levels of Cyp2b2 and Mrp2 in the DEN-PB and DEN-PB-EMIQ groups compared with the DEN-alone group, while the level of Mrp2 decreased in the DEN-PB-EMIQ group compared with the DEN-PB group. There were no significant changes in microsomal reactive oxygen species (ROS) production and oxidative stress markers between the DEN-PB and DEN-PB-EMIQ groups. Immunohistochemically, the constitutive active/androstane receptor (CAR) in the DEN-PB group was clearly localized in the nuclei, but its immunoreactive intensity was decreased in the DEN-PB-EMIQ group. These results indicate that EMIQ suppressed the liver tumor-promoting activity of PB by inhibiting nuclear translocation of CAR, and not by suppression of oxidative stress.

Preventive effects of calcitriol on the development of capsular invasive carcinomas in a rat two-stage thyroid carcinogenesis model.

We have shown phosphoinositide 3-kinase (PI3K)/Akt signaling activation in thyroid capsular invasive carcinomas (CICs), which are highly induced by promotion with sulfadimethoxine (SDM) in a rat 2-stage thyroid carcinogenesis model. To examine the potency of calcitriol, a synthetic vitamin D3 analog, on the development or progression of CICs, male F344 rats were injected with calcitriol (0.1 µg/kg body weight) three times a week intraperitoneally, during an entire period of SDM-promotion for 13 weeks (Experiment 1) or during the last 2 weeks of a 15-week SDM-promotion (Experiment 2). Initiation with N-bis(2-hydroxypropyl)nitrosamine preceded all treatments. In Experiment 1, long-term calcitriol treatment reduced the multiplicity of CICs, while cell proliferation activity, estimated by Ki-67 cell index in the induced CICs, was unchanged with SDM-promotion alone. Considering the strong dependency of promotion with SDM during the early stages on thyroid-stimulating hormone, the reduced multiplicity in Experiment 1 may be due to the effect on an early stage of neoplastic proliferation. Although the magnitude was mild, cell proliferation activity was decreased in existing CICs after short-term calcitriol treatment in Experiment 2, which was associated with a mild decrease in cyclin-dependent kinase-2-positive cells, cytoplasmic immunolocalization of phosphorylated, inactive, Rb protein and a mild increase in nucleocytoplasmic expression of p27(kip1). Although the effect was mild at the late stage of SDM-promotion in this hypothyroidism-related thyroid carcinogenesis model, our results suggest that calcitriol targets cell proliferation via inhibition of a molecular cascade downstream of PI3K/Akt signaling, controlling G1/S transition.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model.

To clarify the involvement of signaling of transforming growth factor (TGF)-ß during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6weeks after starting promotion) and late-stage promotion (57weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P(+) foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P(+) lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P(+) foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P(-) foci induced by promotion with agonists of peroxisome proliferator-activated receptor-α did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P(+) proliferative lesions.

Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats.

To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor alpha agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats.

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo-keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

Elevation of cell proliferation via generation of reactive oxygen species by piperonyl butoxide contributes to its liver tumor-promoting effects in mice.

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT-PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.